Unknown,Transcriptomics,Genomics,Proteomics

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Haploid and diploid Emiliania huxleyi (RCC 1217 and RCC1216) under P starvation

ABSTRACT: Haploid (1N) and diploid (2N) Emiliania huxley (RCC 1217 and RCC1216) were grown in dilute batch culture and achieved stationary phase. We sampled two time points, at early (T2) and late (T3) stationary phase. Controls are haploid or diploid cells derived from dilute cultures (T1) growing in replete control medium. All samples were hybridized against a common, pooled baseline RNA. All experiments and hybridization were done in independent biological triplicates (A, B and C).

ORGANISM(S): Emiliania huxleyi

SUBMITTER: Sebastian Rokitta

PROVIDER: E-MTAB-3877 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cell growth rate is regulated in response to resource availability including the abundance, and molecular form, of essential nutrients. In the model eukaryotic cell, Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen impacts both cell growth rate and mRNA expression. Disentangling causal relationships between nitrogen availability, cell growth rate and differential gene expression poses a considerable challenge. Using experimental control of cell growth rate using chemostats, we studied the effect of variation in environmental nitrogen on differential gene expression. We find that the primary determinant of nitrogen-regulated gene expression is nitrogen abundance whereas variation in nitrogen source affects the expression of only a small number of transcripts with highly specialized functions. To study the dynamics of nitrogen-responsive gene expression we perturbed steady-state nitrogen-limited chemostat cultures by addition of either proline or glutamine. Addition of either proline or glutamine to cells growing in nitrogen-limited chemostats results in repression of the nitrogen catabolite repression (NCR) regulon consistent with nitrogen abundance, and not nitrogen source, being the primary determinant of nitrogen-regulated gene expression. We find that a transition from nitrogen-limited to nitrogen-replete conditions is accompanied by rapid induction of transcripts required for protein translation. We identified a reciprocal relationship between specific regulons required for protein translation (RP and RiBi) and the NCR regulon. Using mathematical modeling we find evidence that cells adopt a metabolically inefficient growth mode during this transition. By means of high resolution time series analysis we find evidence that rapid, and potentially accelerated, mRNA degradation plays an important role in remodeling gene expression programs in response to change in environmental nitrogen. We propose that the evolutionarily conserved TORC1 signaling pathway orchestrates the balance between protein translation and assimilation of nitrogen sources at the transcriptional level to optimize rates of cell proliferation. A total of of 102 samples were analyzed in different nitrogen-limited conditions using chemostats in both steady-state and dynamic conditions. A common reference obtained from an ammonium-limited chemostat growing at a dilution rate of 0.12/hr was used for all two color hybridization experiments.

Project description:Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and Analysis of Sequence Counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. K-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes. Axenic T. pseudonana CCMP 1335 was grown at 14Â°C under 24 hour light (120 Âµmol photons m-2 s-1) after Dyhrman et al. (2012) in f/2 plus silica chelated media made from surface Sargasso Sea water. Nitrate, silica, vitamins, and trace metals were at f/2 concentrations (Guillard and Ryther 1962), while iron and phosphate were modified across treatments. In brief, triplicate cultures of replete (36 ÂµM PO4, 400 nM Fe), P-limited (0.4 ÂµM PO4, 400 nM Fe), Fe-limited (36 ÂµM PO4, 40 nM Fe), and Co-limited (0.4 ÂµM PO4, 40 nM Fe) treatments were harvested when growth deviated from the replete control. Growth was monitored by cell counts. Biomass was harvested onto 0.2 Âµm filters and flash frozen in liquid nitrogen and total RNA was extracted as described in Dyhrman et al. (2012). Tag-seq sequencing of the transcriptome was performed by Illumina with a polyA selection and NlaIII digestion, resulting in 21 bp sequence reads or tags (Dyhrman et al., 2012). Libraries were of varied sizes as follows: replete (~12 million), P-limited (~13 million), Fe-limited (~23 million), and Co-limited (~26 million). Tags were mapped to gene models (predicted protein coding regions) with a pipeline designed by Genesifter Inc., requiring 100% identity and covering 9759 genes. Tag counts within a gene were pooled and normalized to the size of the library, with resulting data expressed in tags per million (tpm). Genes with normalized tag counts less than 2.5 tpm for each of the four treatments were excluded (Figure S1) , leaving 7380 genes in the analysis.

Project description:One of the central goals of evolutionary biology is to explain and predict the molecular basis of adaptive evolution. We studied the evolution of genetic networks in Saccharomyces cerevisiae (budding yeast) populations propagated for more than 200 generations in different nitrogen-limiting conditions. We find that rapid adaptive evolution in nitrogen-poor environments is dominated by the de novo generation and selection of copy number variants (CNVs), a large fraction of which contain genes encoding specific nitrogen transporters including PUT4, DUR3 and DAL4. The large fitness increases associated with these alleles limits the genetic heterogeneity of adapting populations even in environments with multiple nitrogen sources. Complete identification of acquired point mutations, in individual lineages and entire populations, identified heterogeneity at the level of genetic loci but common themes at the level of functional modules, including genes controlling phosphatidylinositol-3-phosphate metabolism and vacuole biogenesis. Adaptive strategies shared with other nutrient-limited environments point to selection of genetic variation in the TORC1 and Ras/PKA signaling pathways as a general mechanism underlying improved growth in nutrient-limited environments. Within a single population we observed the repeated independent selection of a multi-locus genotype, comprised of the functionally related genes GAT1, MEP2 and LST4. By studying the fitness of individual alleles, and their combination, as well as the evolutionary history of the evolving population, we find that the order in which these mutations are acquired is constrained by epistasis. The identification of repeatedly selected variation at functionally related loci that interact epistatically suggests that gene network polymorphisms (GNPs) may be a frequent outcome of adaptive evolution. Our results provide insight into the mechanistic basis by which cells adapt to nutrient-limited environments and suggest that knowledge of the selective environment and the regulatory mechanisms important for growth and survival in that environment greatly increases the predictability of adaptive evolution. DNA from each evolved clone or population is hybridized vs DNA from the ancestral strain

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Haploid and diploid Emiliania huxleyi (RCC 1217 and RCC1216) under P starvation

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